Cell culture processes have been developed for the growth of single cell bacteria, yeast and molds which are resistant to environmental stresses or are encased with a tough cell wall. Mammalian cell culture, however, is much more complex because such cells are more delicate and have more complex nutrient and other environmental requirements in order to maintain viability and cell growth. Large-scale cultures of bacterial type cells are highly developed and such culture processes are less demanding and are not as difficult to cultivate as mammalian cells. These techniques are highly empirical and a firm theoretical basis is not developed. Bacterial cells can be grown in large volumes of liquid medium and can be vigorously agitated without any significant damage. Mammalian cells, on the other hand, cannot withstand excessive turbulent action without damage to the cells and must be provided with a complex nutrient medium to support growth.
In addition, mammalian cells have other special requirements; in particular most animal cells must attach themselves to some substrate surface to remain viable and to duplicate. On a small scale, mammalian cells have been grown in containers with small microwells to provide surface anchors for the cells. However, cell culture processes for mammalian cells in such microwell containers generally do not provide sufficient surface area to grow mammalian cells on a sufficiently large scale basis for many commercial or research applications. To provide greater surface areas, microcarrier beads have been developed for providing increased surface areas for the cultured cells to attach. Microcarrier beads with attached cultured cells require agitation in a conventional bioreactor vessel to provide suspension of the cells, distribution of fresh nutrients, and removal of metabolic waste products. To obtain agitation, such bioreactor vessels have used internal propellers or movable mechanical agitation devices which are motor driven so that the moving parts within a vessel cause agitation in the fluid medium for the suspension of the microcarrier beads and attached cells. Agitation of mammalian cells, however, subjects them to high degrees of shear stress that can damage the cells and limit ordered assembly of these cells according to cell derived energy. These shear stresses arise when the fluid media has significant relative motion with respect to vessel walls, impellers, or other vessel components. Cells may also be damaged in bioreactor vessels with internal moving parts if the cells or beads with cells attached collide with one another or vessel components.
In addition to the drawbacks of cell damage, bioreactors and other methods of culturing mammalian cells are also very limited in their ability to provide conditions that allow cells to assemble into tissues that simulate the spatial 3-dimensional form of actual tissues in the intact organism. Conventional tissue culture processes limit, for similar reasons, the capacity for cultured tissues to express a highly functionally specialized or differentiated state considered crucial for mammalian cell differentiation and secretion of specialized biologically active molecules of research and pharmaceutical interest. Unlike microorganisms, the cells of higher organisms such as mammals form themselves into high order multicellular tissues. Although the exact mechanisms of this self-assembly are not known, in the cases that have been studied thus far, development of cells into tissues has been found to be dependent on orientation of the cells with respect to each other (the same or different type of cell) or other anchorage substrate and/or the presence or absence of certain substances (factors) such as hormones, autocrines, or paracrines. In summary, no conventional culture process is capable of simultaneously achieving sufficiently low shear stress, sufficient 3-dimensional spatial freedom, and sufficiently long periods for critical cell interactions (with each other or substrates) to allow excellent modeling of in vivo tissue structure.
U.S. Pat. No. 5,155,035, Wolf et al., provides a method that overcomes prior problems without subjecting the cells to destructive amounts of shear, but the production rate of such process is insufficiently low to be of substantial commercial value. The current invention improves on that process by subjecting the culture medium to a time varying electromagnetic force to increase the production rate to commercially significant levels.
A paper entitled: “The Clinostat—A Tool For Analyzing The Influence of Acceleration On Solid-Liquid Systems” by W. Briegleb, published by the proceedings of a workshop on Space biology, Cologne Germany, on Mar. 11, 1983, (ESASP-206, May 1983). In this paper, clinostat principles are described and analyzed relative to gravity affects. Some clinostat experiments are described including experiments where monocellular suspended organisms (protozoans) are placed within cylinders which are rotated about a horizontal axis.
A paper entitled, “The Large-Scale Cultivation of Mammalian Cells”, by Joseph Feder and William R. Tolbert, published in the Scientific American, January 1983, Vol. 248, No. 1. Pgs. 36-43. In this paper, agitation of the cells is described as required to keep the cells suspended in the medium and describes a turbine agitator, a marine propeller agitator, and a vibrating mixer for mixing. The paper also describes a perfusion reactor in which four slowly rotating flexible sheets of monofilament nylon provide agitation The sheets are rotated about a vertical axis while the medium in the main vessel is continuously pumped to the satellite filter vessel. The filter retains the cells which are pumped along with the remainder medium back into the vessel for further proliferation. A paper entitled, “Large Scale Cell Culture Technology”, William R. Tolbert, Joseph Feder, Monsanto Company, St. Louis, Mo., Annual Reports on Fermentation Processes, Vol. 6, 1983, discloses a flat plate hollow fiber system of high culture surface area described as allowing culture of anchorage dependent cells and accumulation of cell secretor products. A culture vessel with flexible spiral vanes for suspension of microcarriers and a fluidized bed with media perfusion is described.
A paper entitled, “Gravisensitivity of the Acellular, Slime, Mold, Physarum, Polycephalum Demonstrated on the Fast Rotating Clinostat”, by Ingrid Block and Wolfgang Brigley, published in the European Journal of Cell Biology 41, Pgs. 44-50, 1986. This paper described rotation of a culture vessel about a horizontal axis for the simulation of weightlessness.
A paper entitled, “Cell and Environment Interactions in Tumor Microregions: The Multicell Spheroid Model”; by Robert M. Sutherland, Science 240: 177-184; (1988) discloses the use of multicell spheroids, without attachment substrates, of tumor cells to study cell and environment interactions in tumors. Conventional culture, processes are utilized to produce limited size and viable tumor cell aggregates.
Cell cultures from various bio-reactors, including a prototype of a slow turning lateral vessel (STLV) designed for batch culturing of cells were presented at a poster session at the First Canadian Workshop on R & D Opportunities on Board the Space Station, National Research Council Canada, May 6-8, 1987, Ottawa, Canada, and published in the Proceedings “Spacebound '87”, as a paper entitled “Growth and Maintenance of Anchorage Dependent Cells in Zero Headspace Bioreactor Systems Designed For Microgravity”, by Lewis et al.
A paper entitled, “Physical Mechanisms of Cell Damage in Microcarrier Cell Culture Bioreactors”, Robert S. Cherry and Eleftherios Terry Poportsakis, Biotechnology and Bioengineering, Vol. 32, Pp. 1001-1014 (1988) discloses mechanisms for damage of anchorage dependent cells cultured on microcarrier beads. Bridging of cells across beads to form bead aggregates is described as a damage mechanism due to breaking of these bridges from mechanical mechanisms.